scholarly journals Thymoquinone-PLGA-PVA Nanoparticles Ameliorate Bleomycin-Induced Pulmonary Fibrosis in Rats via Regulation of Inflammatory Cytokines and iNOS Signaling

Animals ◽  
2019 ◽  
Vol 9 (11) ◽  
pp. 951 ◽  
Author(s):  
Sultan A. M. Saghir ◽  
Naif A. Al-Gabri ◽  
Asmaa F. Khafaga ◽  
Nahla H. El-shaer ◽  
Khaled A. Alhumaidh ◽  
...  
Keyword(s):  
Pulmonary Fibrosis ◽  
Inflammatory Cytokines ◽  
Transforming Growth Factor Beta ◽  
Water Solubility ◽  
Transforming Growth Factor ◽  
Male Rats ◽  
Control Group ◽  
Albino Rats ◽  
The Third ◽  
Alveolar Septa

Pulmonary fibrosis is considered one of the most chronic interstitial illnesses which are not easily treated. thymoquinone’s (TQ) benefits are still partly problematic due to poor water solubility; therefore, it was loaded onto PLGA-PVA carriers. This study aimed to evaluate the potential effect of TQ-PLGA-PVA nanoparticles (TQ-PLGA-PVA-NPs) on pulmonary fibrosis induced by bleomycin in albino rats. Forty male rats were randomized into four groups. The first group served as the control group; the second and the third groups received bleomycin intratracheally, whereas the third group received TQ-PLGA-PVA-NPs after 4 weeks from bleomycin administration. The fourth group was administrated TQ-PLGA-PVA-NPs alone. The designed nanoparticles appeared around 20 nm size (10–30 nm), had a spherical shape, and had 80% encapsulation efficiency. The histological examination of rats simultaneously treated with TQ-PLGA-PVA-NPs and bleomycin revealed reduction in the thickness of the alveolar septa and improvement of the other lung structures, with the presence of lymphocytes admixed with exfoliated epithelium in a few lumina remaining. Ultrastructural findings revealed marked collagenolysis and the release of nanoparticles from ruptured pneumocytes within the alveolar septa after 14 days from TQ-PLGA-PVA-NPs administration. Very active pneumocyte types II were seen in the TQ-PLGA-PVANP group. Additionally, immunohistochemical expression of inducible nitric oxide (iNOS) and estimation of inflammatory cytokines in lung tissues including interleukin 10 (IL 10) and transforming growth factor-beta (TGF-β1) confirmed the antioxidant and anti-inflammatory effects of TQ-PLGA-PVANPs. The study concluded that TQ-PLGA-PVA-NPs could attenuate the bleomycin-induced pulmonary fibrosis, through the inhibition of lung inflammation and the suppression of bleomycin- induced oxidative stress.

Maternal Sodium Valproate Exposure Alters NeuroendocrineCytokines and Oxido-inflammatory Axes in Neonatal Albino Rats

2020 ◽  
Vol 20 ◽  
Author(s):  
Naama A-G ◽  
El-bakry AM ◽  
Rasha EH ◽  
Ahmed RG
Keyword(s):  
Growth Factor ◽  
Sodium Valproate ◽  
Transforming Growth Factor Beta ◽  
Transforming Growth Factor ◽  
Control Group ◽  
Albino Rats ◽  
Serum Growth Hormone ◽  
Pregnant Rats ◽  
Necrosis Factor Alpha ◽  
Insulin Growth Factor

Objective: he aim of the study was to determine the influence of maternal sodium valproate (SVP) on neonatal neuroendocrine (hypothalamic-pituitary-adrenal; HPA)-cytokines and oxido-inflammatory axes. Methods: Pregnant rats (Rattus norvegicus) were orally administered (by gavage) SVP (50 mg/kg) from gestation day (GD) 8 to lactation day (LD) 21. Results: The elevation in serum corticotropin-releasing hormone (CRH), corticosterone, and adrenocorticotropic hormone (ACTH) levels was highly significant at postnatal days (PNDs) 14 and 21 in both dams and neonates of the maternal SVPtreated group relative to those in the control group. However, hypercortisolism (cortisolemia) was highly significant in neonates at both PNDs 14 and 21 while in dams, it was not significantly increased at LD 14 but was at LD 21. This disruption caused adverse effects on maternal food consumption and maternal/neonatal body weight. The maternal SVP treatment resulted in higher levels of neonatal serum adrenaline, noradrenaline, neuropeptide Y (NPY), tumor necrosis factor-alpha (TNF-α), leptin, interleukins (IL-1β, IL-17, IL-4, IL-6 & IL-2), transforming growth factor-beta (TGF-β), and prostaglandin E2 (PGE2), and lower levels of neonatal serum growth hormone (GH), insulin growth factor-1 (IGF-1) and adiponectin at both PNDs. This administration also induced the oxidative stress in neonatal cerebrum and cerebellum at both tested PNDs via the production of free radicals (malondialdehyde; MDA & nitric oxide; NO) and reduction of antioxidant parameters (glutathione; GSH, superoxide dismutase; SOD & catalase; CAT). Conclusion: Maternal SVP treatment stimulated neonatal stress-brain (HPA) axis, resulted in an oxido-inflammatory state, and disrupted the neuroendocrine-cytokines axis, and generally neonatal health.

Protective effects of olmesartan and l-carnitine on doxorubicin-induced cardiotoxicity in rats

2020 ◽  
Vol 98 (4) ◽  
pp. 183-193 ◽  
Author(s):  
Malek M. Aziz ◽  
Mai A. Abd El Fattah ◽  
Kawkab A. Ahmed ◽  
Helmy M. Sayed
Keyword(s):  
Transforming Growth Factor Beta ◽  
Troponin I ◽  
Transforming Growth Factor ◽  
Antineoplastic Agent ◽  
The Other ◽  
Control Group ◽  
Albino Rats ◽  
Protective Effects ◽  
Group I ◽  
Creatine Kinase Mb

Doxorubicin (DOX), an anthracycline antibiotic, is an important antineoplastic agent due to its high antitumor efficacy in hematological as well as in solid malignancies. The clinical use of DOX is limited due to its cardiotoxic effects. The present study aimed to investigate the possible protective effect of olmesartan (Olm), l-carnitine (L-CA), and their combination in cardiotoxicity induced by DOX in rats. Male albino rats were randomly divided into seven experimental groups (n = 8): group I: normal control, group II: L-CA, group III: Olm, group IV: DOX. The other three groups were treated with Olm (10 mg/kg), L-CA (300 mg/kg), and their combination for 2 weeks after induction of cardiotoxicity by a single dose of DOX (20 mg/kg). In the results, DOX showed a significant elevation in serum troponin I, creatine kinase-MB (CK-MB), and lactate dehydrogenase (LDH) together with increased inflammation manifested by the rise of tumor necrosis factor-alpha (TNF-α), intercellular adhesion molecules-1 (ICAM-1), interleukin IL-1β (IL-1β), myeloperoxidase (MPO), nuclear factor-kappa B (NF-κB), and transforming growth factor beta (TGF-β) in cardiac tissues as well as DOX-induced oxidative stress by increasing in malondialdehyde (MDA) and decreasing in superoxide dismutase (SOD) and glutathione (GSH) in heart tissues. In addition, caspase-3 activity was boosted as indication of increased apoptosis. On the other hand, administration of L-CA and Olm attenuated the DOX-evoked disturbances in the abovementioned parameters. In addition, DOX exhibited echocardiographic changes and severe histopathological changes, which were significantly reversed by L-CA and Olm treatment. In conclusion, the present study data confirm the protective role of L-CA and Olm in DOX-induced cardiotoxicity, which may be related to its antioxidant, antiinflammatory, and antiapoptotic agents.

scholarly journals Renoprotective Effect of Platelet-Rich Plasma on Cisplatin-Induced Nephrotoxicity in Rats

2018 ◽  
Vol 2018 ◽  
pp. 1-10 ◽  
Author(s):  
Neveen Salem ◽  
Nawal Helmi ◽  
Naglaa Assaf
Keyword(s):  
Growth Factor ◽  
Transforming Growth Factor Beta ◽  
Transforming Growth Factor ◽  
Platelet Rich Plasma ◽  
Male Rats ◽  
Intercellular Adhesion Molecule ◽  
Control Group ◽  
Protective Effects ◽  
Intercellular Adhesion Molecule 1 ◽  
Histopathological Investigation

Platelet-rich plasma (PRP) has grown as an attractive biologic instrument in regenerative medicine for its powerful healing properties. It is considered as a source of growth factors that may induce tissue repairing and improve fibrosis. This product has proven its efficacy in multiple studies, but its effect on cisplatin-induced nephrotoxicity has not yet been elucidated. The present investigation was performed to estimate the protective impact of platelet-rich plasma against cisplatin- (CP-) evoked nephrotoxicity in male rats. Nephrotoxicity was induced in male Wistar rats by right uninephrectomy followed by CP administration. Uninephrectomized rats were assigned into four groups: (1) control group, (2) PRP group, (3) CP group, and (4) CP + PRP group. PRP was administered by subcapsular renal injection. Renal function, inflammatory cytokines, and growth factor level as well as histopathological investigation were carried out. Treatment with PRP attenuated the severity of CP-induced nephrotoxicity as evidenced by suppressed creatinine, blood urea nitrogen (BUN), and N-acetyl glucosaminidase (NAG) levels. Moreover, PRP depressed intercellular adhesion molecule-1 (ICAM-1), kidney injury molecule-1 (KIM-1), caspase-3, and transforming growth factor-beta 1 (TGF-β1) levels, while enhanced the epidermal growth factor (EGF) level. These biochemical results were reinforced by the histopathological investigation, which revealed restoration of normal renal tissue architectures. These findings highlight evidence for the possible protective effects of PRP in a rat model of CP-induced nephrotoxicity, suggesting a new avenue for using PRP to improve the therapeutic index of cisplatin.

scholarly journals Garlic Alleviates the Injurious Impact of Cyclosporine-A in Male Rats through Modulation of Fibrogenic and Steroidogenic Genes

Animals ◽  
2020 ◽  
Vol 11 (1) ◽  
pp. 64
Author(s):  
Mustafa Shukry ◽  
Saqer S. Alotaibi ◽  
Sarah M. Albogami ◽  
Nora Fathallah ◽  
Foad Farrag ◽  
...  
Keyword(s):  
Gene Expression ◽  
Cyclosporine A ◽  
Transforming Growth Factor ◽  
Substantial Reduction ◽  
Density Lipoprotein ◽  
Male Rats ◽  
Seminiferous Tubules ◽  
Control Group ◽  
Albino Rats ◽  
Cell Degeneration

This work aimed to study the hepato-testicular protective effect of garlic in rats treated with cyclosporine A (CsA). Forty male Westar albino rats were randomly distributed in five groups (8 rats each): control, olive oil, garlic, CsA, and CsA co-treated with garlic. CsA induced an upsurge in the alanine transaminase, aspartate transaminase, and alkaline phosphatase levels and decreased albumin and total protein levels, expression of superoxide dismutase (SOD) gene, serum testosterone, triiodothyronine, and thyroxine levels compared to the control group. Additionally, there was an increase in the cholesterol, triglyceride, and low-density lipoprotein levels and a substantial reduction in the high-density lipoprotein levels compared to the control groups. Histopathological investigation of the liver showed abnormalities like hepatic cell degeneration, congestion of blood vessels, and highly active Kupffer cells in the CsA group. Histopathological examination of testes showed damaged seminiferous tubules, stoppage of the maturation of spermatogonia, and the presence of cells with irregular dense nuclei in the lumina of some tubules. For the groups treated with garlic, mitigation of the damage caused by CsA in the liver and testes, liver function tests, lipid profiles, and hormones was seen along with improved gene expression of SOD and steroidogenesis genes, and decreased gene expression of collagen I-α1 and transforming growth factor-1β. Conclusively, garlic had a positive impact on CsA-induced hepatic and sperm toxicity. It is recommended that garlic should be supplemented in transplant treatments using CsA to alleviate the cyclosporin-induced oxidative injuries and other harmful effects.

scholarly journals EFFECTIVENESS AND MECHANISM OF PHYSICIAN HERBS FROM KALI PUTIH BATUR BANJARNEGARA CENTRAL JAVA AGAINST DIABETIC NEPHROPATHY

2018 ◽  
Vol 11 (12) ◽  
pp. 452
Author(s):  
Kintoko Kintoko ◽  
Hardi Astuti Witasari ◽  
Djati Wulan Kusumo ◽  
Halid Kapri ◽  
Tya Muldiyana ◽  
...  
Keyword(s):  
Diabetic Nephropathy ◽  
Traditional Medicine ◽  
Transforming Growth Factor Beta ◽  
Transforming Growth Factor ◽  
Negative Control ◽  
Male Rats ◽  
Control Group ◽  
Sprague Dawley ◽  
Central Java ◽  
Cox 2

Objectives: Complications in the kidneys (nephropathy) are one of the chronic complications of diabetes mellitus (DM) most common microvascular and estimated to reach 30–40% of all sufferers of DM. Until now there is no cure drug that can prevent diabetic nephropathy. Therefore, the handling of this issue should be done seriously, one of them through an exploration of drug discovery and drug material. Ristoja in 2015 in the ethnic Javanese Banyumasan successfully explores the types of plants, herb, and traditional medicine culture. One is conducted by the Kaliputih Traditional Medicine, Batur, Banjarnegara, Central Java. Based on the results of the interview, traditional medicine has herb for disease therapy kidney failure which consists of 11 species of plants.Methods: The herbs were extracted by infundation method. Sprague Dawley albino male rats were divided into 3 groups (normal, positive, and negative) and 3 sample test groups with 3 different doses (18, 36, and 54 mL/kg body weight [BW]) previously induced streptozotocin. Observations were carried on the levels blood urea nitrogen (BUN), creatinine, uric acid, and nuclear factor kappa B (NF-κB), cyclooxygenase-2 (COX-2), and transforming growth factor-beta (TGF-β) kidney immunohistochemically and histology analysis.Results: Statistical results showed a significant increase of BUN levels in all dose variation groups after being given herbs, compared to the negative control group. The result of the examination of biochemical parameters of creatinine levels statistic showed significant (p<0.05) decrease in the dose 18 and 36 mL/kg BW compare with the negative group. The result of the study on histopathology kidney organs there are are damages to each test in each organ that is necrosis. The result of NF-κB, COX-2, and TGF-β expression no significant decrease compared with the negative controls.Conclusion: The herbs are not capable of nephropathy diabetic and need more research to know that activity as nephroprotective.

scholarly journals Effect of nanopolysaccharide (BSEPS) from Bacillus subtilis sp. on thioacetamide-induced liver fibrosis in rats

2019 ◽  
Vol 43 (1) ◽  
Author(s):  
Manal G. Mahmoud ◽  
Mohsen S. Asker ◽  
Mohamed E. El Awady ◽  
Amal I. Hassan ◽  
Nadia A. R. Zaharan ◽  
...  
Keyword(s):  
Bacillus Subtilis ◽  
Liver Fibrosis ◽  
Transforming Growth Factor Beta ◽  
Transforming Growth Factor ◽  
Periodate Oxidation ◽  
Hepatic Tissue ◽  
Albino Rats ◽  
Gamma Glutamyl Transferase ◽  
Glutamyl Transferase ◽  
Tgf Β1

Abstract Background Nanomedicine contributes to the efficiency of pharmacological treatments and progresses rapidly. The present study was designed to produce exopolysaccharide (BSEPS) from Bacillus subtilis sp. strain reported in our previous study was further characterized, and its BSEPS for synthesis of the nanoparticle Ag-BSEPS using microwave heating to determine the possible effects of a prepared solution containing Ag-BSEPS versus thioacetamide (TAA) evoked liver fibrosis in Wister albino rats. Nanoparticles with silver (Ag) core have been synthesized in an aqueous solution after exposure of BSEPS to periodate oxidation. Animals were split into four groups: I - control rats, water ad libitum for 6 weeks; II - rats were injected with TAA 200 mg/kg-1 3 times/week for 4 weeks IP; III - Ag-BSEPS 100 mg/kg-1 IP twice a week for 6 weeks; and IV - TAA, as group II followed by Ag-BSEPS as group III. The antifibrotic effects of Ag-BSEPS were appraised by determining different hepatotoxicity indices, oxidative stress, and inflammatory and liver fibrosis markers. Results Nanoparticles were obtained with a diameter size range of 50–100 nm characterized by SEM and TEM without using any harmful reagents. Results evinced considerably reduced activity of liver functions such as transaminases (AST, ALT), gamma-glutamyl transferase (GGT), and alkaline phosphatase (ALP) in the group which received TAA followed by Ag-BSEPS compared to the other group which received only TAA. In the current results, the administration of Ag-BSEPS showed an improvement in the proinflammatory cytokines. On the contrary, the antioxidant enzymes in liver homogenates revealed significant improvement (concentration of glutathione peroxidase (GSH-PX), superoxide dismutase (SOD), and catalase (CAT) increases) in animals with TAA-induced liver damage followed by Ag-BSEPS. Moreover, the activities of the fibrotic markers transforming growth factor-beta 1(TGF-β1) and type III pro-collagen (PCIII) were increased in liver tissues in the group which was given TAA alone as compared to the controls. The percentage of fibrosis of hepatic tissue had a positive correlation with the levels of PCIII and TGF-β1, followed by Ag-BSEPS compared to the TAA group without nanocomposite treatment. Microscopic examinations revealed inhibitory effects of Ag-BSEPS on inflammatory changes and deterrent of liver fibrosis. Conclusion It was suggested that the biochemical and histological amelioration observed in Ag-BSEPS (100 mg/kg-1 twice a week for 6 weeks) treated the fibrotic rats.

A Study on the Effect of Samoa Extract (Cleome droserifolia) on Sperm Quality in Male Albino Rats Exposed to Pyrethroid

2021 ◽  
pp. 39-45
Author(s):  
Nura I. Al-Zail ◽  
Salah F. Kamies
Keyword(s):  
Sperm Quality ◽  
Group Iv ◽  
Male Rats ◽  
Control Group ◽  
Albino Rats ◽  
Protective Agent ◽  
Group V ◽  
Group Iii ◽  
Group I ◽  
Induced Changes

Pyrethroid cyhalothrin (PC) is an insecticide that is used worldwide for pest control in agriculture and household use. Samoa extract (SE) is a potent antioxidant protecting cells from oxidative stress. The present study investigates the protective and therapeutic effect of SE on PC-induced changes in sperm quality in male rats. Fifty adult male albino rats were divided into five groups: group I: served as control; group II: received PC i.p. only (6.2 mg/kg b.wt.); group III: received SE only (100 mg/kg b.wt., p.o.) for eight weeks; group IV: received SE as a protective agent daily for eight weeks, then followed by the administration of PC (i.p.) three times a week for two weeks; group V: exposed to PC (i.p.) three times a week for two weeks, then treated with the SE daily for 8 weeks. Results showed that PC caused markedly impaired sperm quality (a count, viability, motility, and abnormality). Compared to PC-treated animals, SE in the protective group markedly restored the alteration of sperm indices. However, SE in the curative group was found to be less effective in restoring PC-induced alterations. In conclusion, the data of this study revealed that the SE as a protective agent is more effective than as a therapeutic agent. Keywords: Samoa; Pyrethroid; Sperm quality; Rat

scholarly journals A Rnd3/p190RhoGAP pathway regulates RhoA activity in idiopathic pulmonary fibrosis fibroblasts

2018 ◽  
Vol 29 (18) ◽  
pp. 2165-2175 ◽  
Author(s):  
Elizabeth Monaghan-Benson ◽  
Erika S. Wittchen ◽  
Claire M. Doerschuk ◽  
Keith Burridge
Keyword(s):  
Extracellular Matrix ◽  
Idiopathic Pulmonary Fibrosis ◽  
Pulmonary Fibrosis ◽  
Transforming Growth Factor Beta ◽  
Transforming Growth Factor ◽  
Normal Lung ◽  
Rhoa Activity ◽  
Incurable Disease ◽  
Normal Lung Function ◽  
Growth Factor Beta

Idiopathic pulmonary fibrosis (IPF) is an incurable disease of the lung that is characterized by excessive deposition of extracellular matrix (ECM), resulting in disruption of normal lung function. The signals regulating fibrosis include both transforming growth factor beta (TGF-β) and tissue rigidity and a major signaling pathway implicated in fibrosis involves activation of the GTPase RhoA. During studies exploring how elevated RhoA activity is sustained in IPF, we discovered that not only is RhoA activated by profibrotic stimuli but also that the expression of Rnd3, a major antagonist of RhoA activity, and the activity of p190RhoGAP (p190), a Rnd3 effector, are both suppressed in IPF fibroblasts. Restoration of Rnd3 levels in IPF fibroblasts results in an increase in p190 activity, a decrease in RhoA activity and a decrease in the overall fibrotic phenotype. We also find that treatment with IPF drugs nintedanib and pirfenidone decreases the fibrotic phenotype and RhoA activity through up-regulation of Rnd3 expression and p190 activity. These data provide evidence for a pathway in IPF where fibroblasts down-regulate Rnd3 levels and p190 activity to enhance RhoA activity and drive the fibrotic phenotype.

scholarly journals Effect of Tramadol on Sperm Profile of Male Albino Rats

2019 ◽  
pp. 1-6
Author(s):  
I. S. Esua ◽  
U. U. Uno ◽  
U. B. Ekaluo
Keyword(s):  
Sperm Motility ◽  
Sperm Count ◽  
Sperm Head ◽  
Male Rats ◽  
Control Group ◽  
Albino Rats ◽  
Dependent Manner ◽  
Sperm Viability ◽  
Albino Rat ◽  
Significant Difference

Background and Aim: Tramadol is a potent analgesic effective in the treatment of mild to severe pains. However, the use of the drug can pose a threat to other organs and systems. Therefore, this study evaluated the effect of graded doses of tramadol on sperm profile of male albino rats. Materials and Methods: Eighteen male rats were divided into three groups (A, B and C) using completely randomized design (CRD) with six rats in each group. Rats in group A served as the control group and were given just food and water while groups B and C were given tramadol at 50 and 100 mg/kg body weight (BW) respectively, daily for the period of 65 days. The treatment was administered via oral gavage and at the end of the treatments, the rats were sacrificed. Immediately after sacrifice, a puncture was made in the epididymis with a sterile pin and examined for semen pH. The epididymes were processed for epididymal sperm motility, viability, count and sperm head abnormality. Results: There was no significant difference in the weight of testes and semen pH. Sperm viability, sperm motility, sperm count and weight of epididymes significantly reduced (p<0.05) in tramadol treated animals when compared with the control. Results also indicated statistically significant (p<0.05) increase in sperm head abnormalities in rats treated with tramadol when compared with the control. Conclusion: The results obtained from this study reveal that tramadol has negative effects on weight of epididymes, sperm count, sperm viability, sperm motility and sperm head abnormalities in male albino rat as mammalian models in a dose dependent manner.

scholarly journals Di-Tyrosine Crosslinking and NOX4 Expression as Oxidative Pathological Markers in the Lungs of Patients with Idiopathic Pulmonary Fibrosis

Antioxidants ◽  
2021 ◽  
Vol 10 (11) ◽  
pp. 1833
Author(s):  
Sanja Blaskovic ◽  
Yves Donati ◽  
Isabelle Ruchonnet-Metrailler ◽  
Tamara Seredenina ◽  
Karl-Heinz Krause ◽  
...  
Keyword(s):  
Idiopathic Pulmonary Fibrosis ◽  
Pulmonary Fibrosis ◽  
Transforming Growth Factor Beta ◽  
Transforming Growth Factor ◽  
Human Fibroblasts ◽  
Cell Types ◽  
Post Translational Modification ◽  
Pulmonary Tissue ◽  
Nadph Oxidase 4

Idiopathic pulmonary fibrosis (IPF) is a noninflammatory progressive lung disease. Oxidative damage is a hallmark of IPF, but the sources and consequences of oxidant generation in the lungs are unclear. In this study, we addressed the link between the H2O2-generating enzyme NADPH oxidase 4 (NOX4) and di-tyrosine (DT), an oxidative post-translational modification in IPF lungs. We performed immunohistochemical staining for DT and NOX4 in pulmonary tissue from patients with IPF and controls using validated antibodies. In the healthy lung, DT showed little or no staining and NOX4 was mostly present in normal vascular endothelium. On the other hand, both markers were detected in several cell types in the IPF patients, including vascular smooth muscle cells and epithelium (bronchial cells and epithelial cells type II). The link between NOX4 and DT was addressed in human fibroblasts deficient for NOX4 activity (mutation in the CYBA gene). Induction of NOX4 by Transforming growth factor beta 1 (TGFβ1) in fibroblasts led to moderate DT staining after the addition of a heme-containing peroxidase in control cells but not in the fibroblasts deficient for NOX4 activity. Our data indicate that DT is a histological marker of IPF and that NOX4 can generate a sufficient amount of H2O2 for DT formation in vitro.

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